Current methods for identifying such compounds are very slow, labor intensive and expensive.

Neurite outgrowth assays are typically performed using cultured neuronal cells, e. g. rat adrenal pheochromocytoma (PC-12) cells which are induced to differentiate when grown in the presence of growth factors, such as nerve growth factor (NGF) -containing media. The cells can be grown in vitro and tested with a variety of compounds and/or with compounds at many different concentrations. However, the existing methods to score neurite outgrowth are labor intensive and time consuming, making high throughput assay development difficult. The manual scoring methods include treating PC-12 cells with a compound of interest, fixing the cells and counting the number of cells per field and visually determining which cells, if any, have neurites. Neurites are qualitatively scored as positive if they are at least twice as long as the cell body diameter. A way to quantitatively measure neurite outgrowth by manual scoring involves tracing the neurites and cell bodies using MCID Elite image analysis software. The number of cells per field is counted. Calculations may then be performed to determine neurite length, number of neurites, total neurite length per cell and mean neurite length per cell in replicates of each concentration.

Imaging Research, Inc of Ontario Canada has developed a machine which automatically scores neurite outgrowth called IN Cell Analyzer 1000. The method of preparing cells for this machine includes steps to stain fixed cells with fluorescein labeled antibody to neurofilament 100 protein or DM1A antibody to tubulin for four hours and then acquire images at a rate of 3.5 min/96 well plate using the IN Cell analyzer. Tubulin and neurofilament are proteins expressed in neurites and cell bodies as well as undifferentiated cells. Drawbacks of this system include a very high price (approximately one million dollars)

for the machine and poor detection in very sparsely covered wells. The machine overestimates neurite length in wells that are sparsely populated due to toxic treatment at high levels of compound. Current methods of measuring neurite outgrowth do not lend themselves to automated screening because of the step of manually measuring cells and neurites either at the data collection step or in order to calibrate imaging equipment.

What are needed in the art are fast, easy, reliable and inexpensive methods for quantifying neurite outgrowth. Such methods are critical in the development of high throughput screening for drug discovery and more timely identification of compounds useful in neurodegenerative diseases.

SUMMARY OF THE INVENTION This invention provides methods for determining the amount of neurite outgrowth in a population of neuronal cells. The invention provides a biomarker (annexin II) that is indicative of neurite outgrowth. Methods of the invention are very sensitive and allow small amounts of neurite outgrowth to be detected. For example, a small increase in annexin II expression can be used as an indicator of neurite outgrowth. In contrast manual observation of neurite outgrowth is not very sensitive since typical assays usually only score cells with neurites that are at least one or one and one half the length of the cell. Accordingly, in some embodiments, the invention provides methods for detecting the outgrowth of neurites that are less than one times the length of the cells being assayed. The invention is also useful for detecting the outgrowth of longer neurites.

In some embodiments, methods include the steps of : (i) detecting annexin II expression in a population of neuronal cells with a detection reagent capable of detecting annexin II ; and (ii) measuring the level of the detection reagent, wherein the amount of annexin II expression indicates the neurite outgrowth of the cells. The invention further provides methods including the step of providing a cell population that includes neuronal cells and incubating the cell population in a cell growth medium prior to detection of annexin II expression.

In one aspect, the invention relates to identifying compounds that increase neurite outgrowth by assaying compounds for their ability to increase annexin II expression, for example, relative to annexin II expression in the absence of any compound or growth factor, or relative to annexin II expression in the presence of a low level of a control compound such as

NGF or other growth factor. Preferred growth factor reference amounts include, but are not limited to, 2 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml, 30 ng/ml, 40 ng/ml and 50 ng/ml. In some embodiments the EC50, EC60, EC70, EC80, or EC90 of a compound can be identified.

Embodiments of the present invention particularly useful for screening test compounds include methods wherein the population of neuronal cells includes a one or more different target cell populations. Useful cell populations for assays of the invention include endothelial cells, tumor cells, monocytes, macrophages, and combinations of thereof. Particularly useful cell populations include neuronal cells.

In another aspect, the present invention provides high-throughput assay methods for rapidly identifying a compound capable of modulating neurite outgrowth of a population of neuronal cells.

In another aspect, the invention provides one or more antibodies against all or an immunogenic portion of annexin II, wherein the antibody is capable of specifically forming an immune complex with annexin II.

In another aspect, the invention also provides a method of detecting annexin II in a biological sample; the method including the steps of : (a) contacting a biological sample with an annexin II detection reagent (e. g. antibody, nucleic acid, or other binding reagent), and detecting the amount reagent bound to annexin II (e. g an amount of an annexin II immune complex), wherein the amount of the annexin II bound reagent is indicative of the amount of neurite outgrowth in the biological sample. In some embodiments, this method involves detecting a signal and comparing it to a reference signal. Methods for detecting annexin II bound reagent can involve one or more wash steps to remove unbound reagent or annexin II, depending on whether annexin II or the reagent is bound to a solid support. Either the reagent or the annexin II can be labeled, or both (preferably with different labels) can be labeled. In other embodiments, the annexin II reagent binding is detected directly in solution. In some embodiments, the cells are lysed. In other embodiments, the cells are not lysed.

As used herein, the term"neurite outgrowth"includes dendritic and axonal outgrowth from neuronal cells. During neurite outgrowth, many proteins are expressed which correspond to cell growth in general.

Besides substantially full-length annexin II, the present invention provides antigenic fragments of annexin II. As used herein, the term"fragment", as applied to annexin II, will ordinarily be about 5 contiguous amino acids and will preferably be at least 10 or at least 20 contiguous amino acids. Such fragments may be included in larger polypeptides provided that the non-annexin II amino acid sequences do not destroy the annexin II peptide antigenic or immunogenic properties. Multiple annexin II fragments may be combined in a single polypeptide or a single mixture of polypeptides to generate antibodies or other annexin II binding reagents.

Useful antibodies are specific for annexin II and are capable of forming a specific annexin II immune complex. By the term"capable of forming a specific immune complex"is meant an antibody that does not substantially bind other molecules. Such an antibody may specifically bind to one or a few other molecules (e. g. related annexin proteins or peptides) provided that they are sufficiently specific to be useful for detecting annexin II. The required specificity will depend on the configuration of the assay.

The presence of annexin II can also be measured in a coagulation assay such as a standard coagulation assay. Reutelingsperger et al. (Eur. J. Biochem. 151: 625,1985, hereby incorporated by reference) describe a modified prothrombin time test which is suitable for determining the coagulation-inhibiting activity of annexin II and fragments thereof.

In another aspect, the invention provides a method for evaluating a treatment for promoting neurite outgrowth. In some embodiments, the method involves administering to a subject suffering from a neurological disease, disorder, or injury, a compound that was identified in an annexin II assay as promoting neurite outgrowth, and monitoring the subject for signs of improvement or recovery.

In other aspects, the invention provides annexin II detection kits including annexin II detection reagents and instructions for detecting and measuring neurite outgrwoth. In yet other embodiments, the invention provides therapeutic compounds and compositions for promoting or decreasing neurite outgrowth.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the effect of an NGF dose curve on neurite outgrowth; Figure 2 illustrates the effect of an NGF dose curve on annexin II expression; Figure 3 illustrates neurite outgrowth in response to treatment with 50 ng/ml NGF in a time-course; Figure 4 illustrates annexin II expression in response to treatment with 50 ng/ml NGF in a time-course; Figure 5 illustrates annexin II expression in response to Y27632 at different concentrations (as a percentage of annexin II expression in response to NGF at a concentration of 50 ng/ml); and Figure 6 illustrates annexin II expression in response to Y27632 and nicotine at different concentrations.

DETAILED DESCRIPTION OF THE INVENTION The invention provides methods, compositions, and kits for assaying neurite outgrowth in cells. The invention relates, in part, to the use of annexin II expression as a quantitative marker of neurite outgrowth. The invention provides both in vitro and in vivo methods for assaying neurite outgrowth. The invention is useful for assaying neurite outgrowth in a high throughput format. The invention is particularly useful for screening compounds to identify candidates that promote neurite outgrowth. The invention is therefore useful for drug discovery. The invention can also be used for evaluating the neurite-outgrowth promoting activity of a compound in vivo. In addition, the invention provides quality control, diagnostic, and therapeutic methods and compositions relating to neurite outgrowth. In some embodiments, the invention provides high throughput assays and kits for screening large numbers of test compounds to identify potential drug candidates that modulate neurite outgrowth using quantitative detection methods and reagents described herein.

The invention is based, in part, on the surprising discovery that the amount of annexin II expression in neuronal cells is strongly correlated with the amount of neurite outgrowth in those cells. Annexin II expression can therefore be used as a marker for qualitative and/or quantitative measurements of neurite outgrowth. The process of neural differentiation is a complex multistage process involving cell signaling pathways and the expression of many different genes. At least 600 differentially expressed messenger RNAs have been associated with the process of NGF induced neuronal differentiation including neuritogenesis (Lee et al.

(1995), Proc. Natl. Acad. Sci. USA, 92,8303-8307). However, according to the invention, expression of annexin II is specifically increased in neuronal cells upon addition of NGF which induces neuronal cell differentiation leading to neurite outgrowth.

Neuritogenesis is a complex process that involves interactions between the growing neurite and the extracellular matrix. Neurite extension involves proteolytic activity and implicates factors such as tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). According to the invention, the extracellular matrix has to be removed by proteases before new neurites can further extend out. According to the invention, Annexin II can act as a co-receptor for t-PA.

The present invention therefore provides methods for determining the amount of neurite outgrowth in neuronal cells by measuring the amount of annexin II expression in the cells. According to the invention, the amount of annexin II can be correlated to the amount of neurite outgrowth. Preferred measurements of neurite outgrowth include, but are not limited to: the number of neurites per cell or per cell population, the average length of neurites on neuronal cells, the average number of long neurites per cell, or the average number of cells which bear long neurites (e. g. one or one and a half times the cell body length). An example of a reference amount of neurite outgrowth is provided by NGF incubation at 50 ng/ml for 5 days which induces 50-60% neurite formation in certain neuronal cell populations.

According to the invention, the amount of annexin II expression can be measured by any method that detects the level of an annexin II nucleic acid or polypeptide (including functional assays for the presence of the polypeptide).

Annexin II Annexin II is a member of the annexins (or lipocortins) family of proteins which bind to negatively charged phospholipids in a calcium-dependent manner. They are found in a variety of cell types in higher and lower eukaryotes. The annexin family of calcium binding proteins shows features of both soluble and membrane proteins. The primary sequences of the 13 known annexin family members are largely composed of four highly conserved homologous repeats. In contrast to the bulk of the protein, the N termini of the annexins differ greatly in length and sequence (Crompton et al., Cell 55: 1-3,1988 ; Barton et al. , Eur. J.

Biochem. 198: 749-760,1991).

The in vivo role of the annexins is unknown. One proposed function is involvement in membrane-membrane fusion and exocytosis (Creutz, Science 258: 924-931,1992). In addition, roles for annexins have been proposed involving interactions with cytoskeletal proteins (Mangeat, Biol. Cell. 64: 261-281), anticoagulant activities (Tait et al. , Biochemistry 27: 6268-<BR> 6276), inhibition of phospholipase A2 in the regulation of inflammation (Davidson et al. , J.

Biol. Chem. 265: 5602-5609,1990), and the formation of calcium selective ion channels in phospholipid bilayers by annexin V and VII (Pollard et al. , Proc. Natl. Acad. Sci. USA<BR> 85: 2974-2978,1988 ; Rojas et al. , J. Biol. Chem. 265: 21207-21215,1990 ; Karshikov et al., Eur. Biophys. J. 20: 337-344,1992). Some members of the annexin family are expressed in a growth-dependent manner (Schlaepfer and Haigler, J. Cell. Biol. 111 : 229-238, 1990) and are targets for cellular kinases in vivo (Moss et al., Novel Calcium Binding Proteins (Claus W.

Heizmann, Ed. ) Springer, Berlin, 1991; Erikson and Erikson, Cell 21: 829-836,1980).

Annexin II has been identified as a co-receptor for plasminogen and tissue plasminogen activator that promotes and localizes plasmin generation near the cell surface of nerve growth factor treated PC-12 cells. Recently it has been reported that neurite outgrowth in vitro requires annexin II-mediated plasmin generation (Jacovina et al (2001) J ; Biol. Chem., 276, 49350-49358). However, annexin II was not previously identified as a useful marker for measuring the amount of neurite outgrowth in neuronal cells.

Annexin II is generally a heterotetramer that contains two approximately 36 kD annexin II (p36) subunits and two approximately 11 kD SlOOA10 (pl 1) subunits. The annexin II heterotetramer can be intracellular, membrane-bound (e. g. on the surface of a cell), or

extracellular. Accordingly, methods of the invention can involve detecting intracellular, extracellular, or membrane-bound annexin II or a combination thereof.

As used herein"annexin II"includes all alternatively spliced forms of one or both annexin II subunits encoded by the same genomic loci. As used herein, an annexin II protein can refer to a heterotetramer, heterotrimer, heterodimer, dimer or monomer of one or both annexin II subunits or antigenic or functional fragments thereof. Annexin II is preferably mammalian annexin II. In some embodiments, mammalian annexin II is cat, dog, rat, mouse, horse, donkey, cow, sheep, goat, or pig annexin II. In preferred embodiments, mammalian annexin II is human annexin II.

According to the invention, annexin II expression is a bio-marker for neurite outgrowth.

Therefore, methods for detecting and measuring annexin II expression are useful for monitoring and quantifying neurite outgrowth.

Detecting and Measuring Annexin II Expression Techniques and detection reagents useful for detecting protein expression are well known in the art. As used herein"expression"of a protein encompasses the presence of nucleic acid molecules, e. g. mRNA transcribed from a gene of interest, and the presence of the protein itself. Protein expression may be identified by a variety of techniques including, but not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques, including membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein molecules.

Nucleic Acid Detection The presence of nucleic acid molecules encoding annexin II can be detected in a biological sample, a cell culture, or in vivo, using annexin II detection reagents useful for DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments of nucleic acid molecules encoding one or more annexin II subunits or fragments thereof. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequence of genes encoding annexin II to detect neuronal cells expressing RNA encoding annexin II. Preferred oligonucleotides for amplification or hybridization are between about 10 and about 100 nucleotides long, and preferably between about 20 and about 80 nucleotides

long, for example about 20, about 30, about 40, about 50, about 60, about 70 or about 80 nucleotides long. In some embodiments, one or more oligonucleotides are complementary to an annexin II mRNA. According to the invention, oligonucleotides include natural and synthetic nucleic acid molecules and modified nucleic acids. The backbone can be phosphodiester, phosphorothioate, peptide-based (e. g. PNA) or any other backbone. The oligonucleotide preferably contains a length of sequence that is at least 80%, preferably more than 90%, preferably 100% identical to an annexin II target sequence. This length can be about 5,10, 15,20, 25,30, 35,40, 45,50 or more bases long. Useful annexin target sequences include annexin II gene and mRNA sequences such as those provided in SEQ ID NO: 1 (p36 subunit of human annexin II) and in SEQ ID NO: 3 (p 11 subunit of human annexin II) or one or more variants thereof. The target sequences can be the entire sequences shown in SEQ ID NOS: 1 and 3, or complementary sequences thereof, or fragments of any one of the above (e. g. corresponding to the oligonucleotides described above). Sequences from other species also can be used.

A wide variety of labels and conjugation techniques are known to those skilled in the art and may be used in various nucleic acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding annexin II include, but are not limited to, oligo-labeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, the sequences encoding annexin II, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commercially available <BR> <BR> kits, such as those provided by Pharmacia & Upjohn (Kalamazoo, Minn. ), Promega (Madison,<BR> Wis. ), and U. S. Biochemical Corp. (Cleveland, Ohio). Suitable reporter molecules or labels which may be used for ease of detection include, but are not limited to, radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Protein Detection The detection of annexin proteins can be accomplished by any of a number of methods using annexin II detection reagents that bind to annexin II protein or one or more subunits or

fragments thereof (e. g. the human p36 peptide shown in SEQ ID NO: 2 or a variant thereof or a fragment of any of the above, the human pl 1 peptide shown in SEQ ID NO: 4 or a variant thereof or a fragment of any of the above, or annexin II peptides from other species). Preferred methods for the detection of annexin II proteins can involve, for example, immunoassays wherein annexin II proteins are detected by their interaction with an annexin II specific antibody. Antibodies useful in the present invention can be used to quantitatively or qualitatively detect the presence of annexin II or antigenic fragments thereof. In addition, reagents other than antibodies, such as, for example, polypeptides that bind specifically to annexin II proteins can be used in assays to detect the level of annexin II protein expression.

Alternatively, detection of annexin proteins may be accomplished by detection and measurement of levels of biological properties associated with annexin proteins, such as for example, phopholipase A, or anticoagulant activity.

Immunoassays useful in the practice of the invention include but are not limited to assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. These assays can be performed under denaturing conditions, non-denaturing conditions, conditions that maintain intact cells, and conditions that result in cell lysis.

Accordingly, detection reagents for measuring annexin II levels include but are not limited to 1) antibodies immunoreactive with annexin II polypeptide or antigenic fragments thereof and 2) annexin II protein or fragments thereof (preferably labeled). Other binding reagents such as anti-sense peptides, which bind to epitopes within the annexin II protein are also useful. Useful detection reagents (including antibodies) can bind to an annexin II antigen with a dissociation constant of between about lu. M and about 1nM (e. g. about lnM, about 10, or about 100 nM). However, the dissociation constant can be lower or higher. In some embodiments, an antibody can have a dissociation constant lower than 1 nM.

Antibodies of the present invention include polyclonal and monoclonal antibodies, as well as antibody fragments and derivatives that contain the relevant antigen binding domain of the antibodies. Antibody preparations that consists essentially of pooled monoclonal

antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are publicly available e. g. rabbit anti-annexin II polyclonal antibody is available from US Biological, catalog number A2295-21 and mouse monoclonal annexin II antibody is available from Transduction laboratories, BD Biosciences as catalog number 610068. Rabbit anti human PI 1 can also be obtained from US Biological (PO box 261, Swampscott, MA 01907). Antibody detection reagents include, but are not limited to, antibodies that bind to a denatured annexin II protein or portion thereof and antibodies that bind to a folded annexin II protein or portion thereof. Polyclonal or monoclonal antibodies to folded annexin II can be made for example by injecting an animal (e. g. a mouse) with a human annexin II heterotetramer, a human annexin II p36 subunit, or a human annexin II pi 1 subunit, or a combination thereof, along with incomplete Freund's adjuvant. Similarly, antibodies can be made against annexin II proteins from other species. The annexin II protein can be recombinant or purified from non-recombinant cells.

The term"antibody"as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F (ab') 2, and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and methods for making antibodies or fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, N. Y. (1988), incorporated herein by reference).

A variety of techniques for detecting and measuring the expression of annexin II, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art.

Examples of such techniques include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), "sandwich"-assays, and fluorescence activated<BR> cell sorting (FACS). These and other assays are well described in the art. (See, e. g. , Hampton,<BR> R. et al. (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn. , Section IV; and Maddox, D. E. et al. (1983) J. Exp. Med. 158: 1211-1216).

Immunoassay formats can employ labeled antibodies to facilitate detection. RIAs have the advantages of simplicity, sensitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kinetics. However, radioactive assays have several disadvantages including a short shelf-life due to radioactive decay, a requirement for special handling and disposal, and a requirement for complex and expensive

analytical equipment. RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T. S. , et al. , North Holland Publishing Company, NY (1978), with particular reference to the chapter entitled"An Introduction to Radioimmune Assay and Related Techniques"by Chard, T. , incorporated by reference herein.

ELISAs have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, such that a large number of detection strategies-- colorimetric, pH, gas evolution, etc.--can be used to quantitate the assay. In addition, the enzyme reagents have relatively long shelf-lives, and lack the risk of radiation contamination that attends to RIA use. ELISAs are described in ELISA and Other Solid Phase Immunoassays (Kemeny, D. M. et al. , Eds. ), John Wiley & Sons, NY (1988), incorporated by reference herein. For these reasons, enzyme labels are particularly preferred for use in an enzyme immunoassay (EIA) (Voller, A. ,"The Enzyme Linked Immunosorbent Assay (ELISA) ", 1978, Diagnostic Horizons 2: 1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.; Voller, A. , et al. , 1978, J. Clin. Pathol. 31: 507-520; Butler, J. E., 1981, Meth. Enzymol.

73: 482-523). The enzyme that is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorimetric, or by visual means. Enzymes that can be used to detectably label the antibody include, but are not limited to, horseradish peroxidase and alkaline phosphatase. Detection can also be accomplished by colorimetric methods that employ a chromogenic substrate for the enzyme.

No single enzyme is ideal for use as a label in every conceivable immunometric assay.

Instead, one must determine which enzyme is suitable for a particular assay system. Criteria important for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time), purity of the enzyme preparation, sensitivity of detection of its product, ease and speed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and its conjugate, and the like. Examples of suitable enzymes include, but are not limited to, peroxidase, acetylcholine esterase, alpha-glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, beta-galactosidase, catalase, delta-5-steroid isomerase, glucose oxidase, glucose- 6-phosphate dehydrogenase, glucoamylase, glycoamylase, luciferase, malate dehydrogenase, peroxidase, ribonuclease, staphylococcal nuclease, triose phosphate isomerase, urease, yeast-

alcohol dehydrogenase, etc. Peroxidase and urease are among the more preferred enzyme labels, particularly because of chromogenic pH indicators which make its activity readily visible to the naked eye.

In lieu of such enzyme labels, chemiluminescent, radioisotopic, or fluorescent. labels may be employed for either labeling antibodies immunoreactive with annexin II or annexin II protein itself (i. e. for competition assays in which labeled annexin II is mixed with cell lysates) or peptides or other reagents that bind to annexin II. Examples of suitable radioisotopic labels include, but are not limited to, 3H,"'In, 1251, 1311, 32p, 35s, 14C, 5'Cr, 57 To, 58Co, 59Fe, 75 Se, l52EU, 90Y, 67CU, 217Ci, 211At, 212Pb, 47SC, l09Pd, etc. Examples of suitable chemiluminescent labels include, but are not limited to, luminal labels, isoluminal labels, aromatic acridinium ester labels, imidazole labels, acridinium salt labels, oxalate ester labels, luciferin labels, aequorin labels, etc. Examples of suitable fluorescent labels include, but are not limited to, fluorescein labels, isothiocyanate labels, rhodamine labels, phycoerythrin labels, phycocyanin labels, allophycocyanin labels, o-phthaldehyde labels, fluorescamine labels, etc. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin and fluorescamine. Likewise, a bioluminescent compound may be used to label the annexin antibody. The presence of a bioluminescence protein is determined by detecting the presence of luminescence. Important bioluminescence compounds for purposes of labeling are luciferin, luciferase and aequorin.

In methods of the invention, a solid phase, e. g. a conventional ELISA plate, is coated with detection reagents such as anti-annexin II antibodies. For antibodies, the solid phase is usually poly (vinyl-chloride), but may be other polymers such as cellulose, polyacrylamide, nylon, polystyrene or polypropylene. The solid supports may be in the form of tubes, beads, discs or micro plates, or any other surfaces suitable for conducting an assay.

In some embodiments of the invention, the levels of annexin proteins can be analyzed by two-dimensional gel electrophoresis. Methods of two-dimensional electrophoresis are known to those skilled in the art. Samples, such as cells samples or tissue samples, are loaded onto electrophoretic gels for isoelectric focusing separation in the first dimension which separates proteins based on charge. A number of first-dimension gel preparations may be utilized including tube gels for carrier ampholytes-based separations or gels strips for immobilized gradients based separations. After first-dimension separation, proteins are

transferred onto the second dimension gel, following an equilibration procedure and separated using SDS PAGE to separate the proteins based on molecular weight. When comparing biological samples derived from different experimental conditions or subjects, multiple gels are prepared for the different samples (including samples from control conditions or subjects).

Following separation, the proteins are transferred from the two-dimensional gels onto membranes commonly used for Western blotting. The techniques of Western blotting and subsequent visualization of proteins are also well known in the art (Sambrook et al,"Molecular Cloning, A Laboratory Manual", 2. sup. nd Edition, Volume 3, 1989, Cold Spring Harbor).

Standard procedures may be used, or the procedures may be modified as known in the art for identification of proteins of particular types, such as highly basic or acidic, or lipid soluble, etc.

(See for example, Ausubel, et al. , 1999, Current Protocols in Molecular Biology, Wiley &<BR> Sons, Inc. , N. Y. ). In some embodiments, unlabeled antibodies that bind to the annexin II proteins are utilized in an incubation step, as in the procedure for a Western blot analysis. A second, preferably labeled, antibody specific for the first antibody is used in the Western blot analysis to visualize proteins that reacted with the first antibody.

According to the invention, annexin II is a useful biomarker for assaying neurite outgrowth. Annexin II assays can be useful to detect neurite outgrowth in many different cells, and test the effectiveness of neurite-inducing or neurite-promoting compounds in many different cells.

Biological Samples Assays of the invention can be performed to detect neurite outgrowth in different cell and tissue types including in vitro cell and tissue cultures, biopsy cell and tissue samples, and in vivo. Populations of target cells of interest are typically contacted with one or more compounds to test their ability to induce neurite outgrowth in the target cell. The invention is particularly useful for-assaying neurite outgrowth in nerve cells and nerve tissue. However, neurite outgrowth can also be assayed in other cells or tissue that can develop neurites, or can be induced to develop neurites. For example, stem cells, pluripotent cells, other neuronal progenitor cells, immortalized cell lines, or cancer cells can be assayed for neurite outgrowth using annexin II detection methods. Target cell populations typically contain one cell type of interest, with possible contamination from other cell types depending on whether the cell population is a cultured cell population or a cell population obtained from a subject.

Generally, at least 10 cells are assayed. However, preferred assays include from about 1,000 to 109 cells for each condition being tested. In some embodiments, the number of cells in each assay reaction is from about 5,000 to about 30,000. For example between about 5,000 and about 30,000 cells can be incubated per well of a 24,96 or 384-well plate in any of the different assay formats described herein. Preferably, the same number of cells are used for each assay reaction (e. g. in each well being assayed on a plate). However, different numbers can be used in some formats. Generally, the number of cells used per reaction is predetermined (e. g. a chosen number of cells are added to each well). However, in some embodiments, the number of cells present in each reaction can be independently measured.

The information concerning the number of cells per reaction is useful to calibrate the results, or to calculate annexin II expression levels or neurite outgrowth and compare data or results from different reactions run in parallel or from different assays run at different times or in different places.

In some embodiments, methods of the invention can be used to identify or assay the activity of compounds that inhibit or reduce neurite outgrowth. Cells or tissue with high levels of neurite outgrowth can be contacted with one or more compounds and a decrease in neurite outgrowth can be detected as a decrease in annexin II expression. Annexin II expression is involved in several biological processes including pathogenic and non-pathogenic conditions.

For example, annexin II expression is involved in tumor cell invasion, angiogenesis, embryogenesis, monocyte and macrophage migration into the sites of inflammation. Diseases involving the above processes include, but are not limited to, various cancers, acute promvelocytic leukemia, atherosclerosis, virus infection, diabetes etc.

Neuronal Cells As used herein, "neuronal cells"means neural cells and their precursors and other cell lines capable of extending neurites. Neuronal cells are preferably isolated for use in assays to evaluate the ability of test compounds to induce neurite outgrowth. Neural cells such as primary neural cells may be isolated as a homogeneous mixture. Examples of neural cell precursors include stem cells and neural progenitor cells which can be directed to differentiate into a neural cell. Neural cells and their precursors often need special culture conditions and may require co-culturing with feeder cells. The generation of other cell lines capable of

extending neurites can be divided into two categories: 1) spontaneously occurring tumors, and 2) custom-designed cell lines.

Of the spontaneously occurring tumor cells, probably the most studied cell line for neurobiology is the rat pheochromocytoma (PC12) cells that can differentiate into sympathetic- like neurons in response to NGF. These cells have proven to be a useful model for studying mechanisms of neural development and alterations (molecular and cellular) in response to growth factors. Neuroblastoma and glioma cell lines have been used to study neuronal and glial functioning [Liles, et al. , J. Neurosci. 7,2556-2563 (1987); Nister et al., Cancer Res.

48 (14) 3910 (1988)]. Neuroblastoma cell lines such as NIE-115, N2A, PC-12 are available from various depositories such as the American Type Culture Collection (ATCC) or the Deutsche Sammlung fur Mikroorganismen (DSM). NG108-15 is a neuroblastoma-glioma hybrid cell line which can undergo morphological (neurite extension) and biochemical (increased choline acetyltransferase) differentiation; these cells can synthesize and store acetylcholine, express neurotransmitter receptors, and form functional synapses with each other and with muscle cells in culture. Embryonal carcinoma cells are derived from teratoma tumors of fetal germ cells and have the ability to differentiate into a large number of non- neural cell types with some lines (e. g. P19 cells) [Jones-Villeneuve et al. J. Cell Biol. 94,253- 262 (1982) ] having the ability to differentiate into neural cells [ (McBumey et al. J. Neurosci.<BR> <P>8 (3) 1063-73 (1993) ]. A human teratocarcinoma-derived cell line, NTera 2/cl. Dl, with a phenotype resembling CNS neuronal precursor cells, can be induced to differentiate in the presence of retinoic acid. However, the differentiated cells are restricted to a neuronal phenotype [Pleasure and Lee J. Neurosci. Res. 35: 585-602 (1993) ]. These types of cell lines are able to generate a large number of cells for screening the effects of exogenous agents on cell survival or neural function and discovery of novel therapeutics.

An alternative approach to spontaneously occurring cell lines is the intentional immortalization of a primary cell by introducing an oncogene that alters the genetic make-up of the cell thereby inducing the cell to proliferate indefinitely. This approach has been used by many groups to generate a number of interesting neural cell lines [ (Bartlett et al. , Proc. Nat.

Acad. Sci. 85 (9) 3255-3259 (1988) ; Frederiksen et al. Neuron 1,439-448 (1988) ; Trotter et al.

Oncogene 4: 457-464 (1989); Ryder et al. , J. Neurobiol. 21: 356-375 (1980); Murphy et al. J.

Neurobiol 22: 522-535 (1991); Almazan and McKay et al. Brain Res. 579: 234-245 (1992)].

These lines are useful for studying the decisions that occur during cell determination and differentiation, and for testing the effects of exogenous agents.

Other cell types suitable for use in methods of the present invention will be apparent to those skilled in the art. These cell types include, but are not limited to, endothelial cell, monocyte, macrophage, neuronal cell or other tumor cell lines. Neuronal cells include, but are not limited to, primary neurons, PC12, N2a, IMR-32, SHSY-5Y or mutants of the above cell lines, such as PC-12D or NeuroScreen (Cellomic), or other neuroblastoma derived cells.

Tissue and Cell Samples In some embodiments, cells are obtained from tissue samples, cell samples, or other biological samples. Aliquots of whole tissues, or cells, can be solubilized using any one of a variety of solubilization cocktails known to those skilled in the art. For example, tissue can be solubilized by addition of lysis buffer comprising (per liter) 8 M urea, 20 ml of Nonidet P-40 surfactant, 20 ml of ampholytes (pH 3.5-10), 20 ml of 2-mecaptoethanol, and 0.2 mM of phenylmetliylsulfonyl fluoride (PMSF) in distilled deionized water.

Immunoassays for detecting expression of annexin II protein typically comprise contacting the biological sample with an anti-annexin antibody under conditions to promote an immunospecific antigen-antibody binding reaction, and detecting or measuring the amount of any immunospecific binding by the antibody or other annexin II binding reagent. The levels of annexin II protein in a biological sample are compared to norms established for cell type and treatment type (e. g. the type and amount of neurite-promoting compounds that the cells were exposed to, or control norms corresponding to the amount of annexin II in the absence of any treatment). The norms can be previously established reference amounts of annexin II or reference amounts established under the experimental conditions (e. g. treatment type) being evaluated.

In some embodiments of the invention, a biological sample such as a tissue extract (or other cell sample described herein) is brought into contact with a solid phase support or carrier, such as nitrocellulose, for the purpose of immobilizing any proteins present in the sample. The support is then washed with suitable buffers followed by treatment with detectably labeled annexin II detection reagent (e. g. an annexin II specific antibody). The solid phase support is then washed with the buffer a second time to remove unbound reagent. The amount of bound reagent on the solid support is then determined according to well known methods.

According to the invention, the amount of annexin II expression can be used as a biomarker to assay the amount of neurite outgrowth induced by one or more test compounds.

Test Compounds Assays of the invention can be used to evaluate one or more test compounds for their ability to induce, promote, or potentiate neurite outgrowth in one or more biological samples described herein. A compound such as NGF can induce neurite outgrowth without any additional co-factors. Some compounds do not effectively induce neurite outgrowth alone.

However, they can potentiate neurite outgrowth induced by other compounds.

Methods of the invention can be used to evaluate the effectiveness of a known inducer, promoter, or potentiator of neurite outgrowth in different cell or tissue types (including cell or tissue types from different individuals). Methods of the invention can also be used to screen libraries of compounds to identify'candidates that induce, promote, or potentiate neurite outgrowth. Methods of the invention can also be used to identify compounds that inhibit or reduce neurite outgrowth. The configuration of the assay can be adapted to the type of compound that is being screened for. In some embodiments, a target cell population is contacted with a single compound. In other embodiments, a target cell population is contacted with two or more compounds, some of which may be known to induce neurite outgrowth. In yet further embodiments, separate groups of cells from a target cell population are separately contacted with each of a plurality of compounds or combinations thereof in order to evaluate the effectiveness of each compound or combination.

Compounds can be tested at any concentration. However, preferred test concentrations range from about 1 nM to about 1 mM. Typical concentrations are between about 1 and about 100 uM. In some embodiments, one or more compounds are tested at a first concentration to screen for active compounds. Active compounds can then be tested at a series of different concentrations to determine their level of activity with greater precision.

Compounds can be incubated with target cells for any amount of time. However, preferred test times range from about 1 hour to about 2 weeks. Typical incubation times are between about 24 hours and about 120 hours. However, shorter or longer times can be used.

In some embodiments, one or more compounds are incubated for a first time period to screen

for active compounds. Active compounds can then be tested by incubation for a series of different time periods to determine their level of activity with greater precision. Compounds can also be added several times during the incubation period (e. g. hourly, two or more times per day, daily, two or more time per week, weekly, or more or less frequently).

Compounds can be incubated with target cells under any growth conditions. However, in most embodiments, growth conditions are optimized for cell growth and development.

Preferred cell culture conditions are those that are similar to in vivo growth conditions. In some embodiments, compounds can be tested by in vivo administration followed by annexin II detection in vivo (e. g. using a labeled annexin II binding reagent the binding of which can be detected non-invasively).

In some embodiments, a test compound is incubated with target cells along with a small amount of one or more known promoters of neurite outgrowth (e. g. NGF or other growth factor such as BGDF, EGF, FGF, IGF etc. , staurosporine, a Rho kinase inhibitor, or other promoters) including naturals product such as picrosides and synthetic products. In other embodiments, the test compound is incubated without any of these promoters. In either format, the effect of the test compound can be compared to the effect of one or more of these promoters in one or more know reference concentrations.

The activity of a test compound can be evaluated by comparing annexin II expression levels induced by the compound to one or more reference annexin II expression levels. A reference annexin II expression level is an amount of annexin II expression obtained for a certain cell population under defined conditions. Defined conditions include: the absence or low level of growth factors, the absence of neurite inducing compounds, the presence of one or more growth factors and/or the presence of one or more neurite inducing compounds.

Examples of reference conditions include, but are not limited to, about 2 ng/ml NGF, about 10 ng/ml NGF, about 20 ng/ml NGF, about 30 ng/ml NGF, about 40 ng/ml NGF, and about 50 ng/ml NGF. The reference amount is preferably obtained in a control assay that is performed along with the test assays. However, the reference amount can be an external reference amount or standard that the results from the test assay are compared to. This type of standard can be used if the assays are reproducible and the signal reagents and reaction conditions are identical or similar in the different assays.

The activity of a test compound can be determined as a relative amount of annexin II expression compared to a known reference. The activity of a test compound can be determined as a relative amount of annexin II expression compared to one or more other test compounds being tested. The relative activity can be a rank (e. g. low, lower, lowest, high, higher, highest, or a rank number relative to other compounds). The relative activity can be a percentage or multiple of a reference, control, or experimental amount. The activity of a test compound can also be determined as an absolute amount, e. g. an amount of annexin II expression or an amount of increase in annexin II expression that results from incubation with the compound.

These amounts can be determined by comparing the intensity of a detection assay signal to the intensity of a signal from a calibration curve generated using known amounts of annexin II.

The activity of a test compound can also be converted into a corresponding amount of neurite outgrowth using known standards for the cells that were contacted with the compound.

Neurite outgrowth can be measured as the average % of cells that bear a neurite longer than the cell body length or diameter (number of cells with a neurite longer than the cell body/total number of cells). Neurite outgrowth can also be measured using other units (including other units described herein.) In some embodiments, the activity of a test compound is compared to a reference or threshold amount to determine whether or not the activity is lower, similar to, or higher than the reference or threshold amount. The threshold amount can be an amount that corresponds to a minimal or maximal level of activity that is acceptable for a particular use. In some embodiments, the threshold can be a cutoff amount for a screen in which only compounds with activities above the threshold amount are selected. In other embodiments, the cutoff is such that only compounds with activities below the threshold are selected. In yet further embodiments, compounds are selected only if they have activities between two threshold amounts. Each threshold amount can correspond to the activity obtained with a reference concentration of a reference neurite outgrowth promoting compound under reference conditions using reference neuronal cells. The assay conditions target cells used to assay a compound are preferably the same as those used to obtain the threshold amount.

Conditions for incubating a test compound with a target cell vary. Incubation conditions will depend on factors such as the type of compound, format, and detection system employed for the assay, as well as the nature of the target cell or tissue used in the assay. For

example, conditions will vary slightly when a whole antibody, a single chain antibody, a F (ab) fragment, or a peptide agent is used. One skilled in the art will recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme- linked immunosorbent assays, diffusion based ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the annexin II detection reagents of the present invention. Examples of such assays can be found in T. Chard, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986) ; G. R. Bullock et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985) ; and P. Tijssen, P. , Practice and Theory of Enzyme Immunoassays : Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

Example 1 describes a neurite outgrowth assay using at least one population of PC-12 cells. Cell growth medium and culture conditions for these and other neuronal cells are known in the art. Each population of neuronal cells is contacted with a test compound. The effect of a test compound on neurite outgrowth as compared to negative and positive controls is then determined using one or more detection reagents described herein. Cell based assays and detection techniques and measurements described herein can be performed using multi-well dishes. High throughput screens are preferably automated and conducted in 384 well dishes.

Test compounds useful in methods of the present invention refers to any agent, such as a virus, protein, peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide, drug, pro- drug or other substance that may have an effect on neural cells whether such effect is harmful, beneficial, or otherwise. Of particular interest are test compounds having a molecular weight of less than 5000 atomic mass units.

Modulators of neurite outgrowth can be identified by comparison of test compounds to positive or negative controls. As used herein"modulate"encompasses the ability to enhancement, inhibition or direct neurite outgrowth. A change in neurite outgrowth is "enhanced"by a test compound if such an effect does not occur in the absence of the test compound (negative control). Test compounds that are beneficial to neural cells e. g. by enhancing neurite outgrowth are referred to herein as"neurological agents", a term which encompasses any biologically or pharmaceutically active substance that may prove potentially useful for the proliferation, differentiation or functioning of CNS cells or treatment of

neurological disease or disorder such as Alzheimer's Disease. For example, the term neurological agents may encompass certain neurotransmitters, neurotransmitter receptors, growth factors, growth factor receptors, low molecular weight organic molecules and the like, as well as enzymes used in the synthesis of these agents.

The term"contacting"refers to exposing the neuronal cell to a test compound so that the test compound can effectively induce neurite outgrowth from the cell. Contacting may be in vitro, for example by adding test compound to a tissue culture of neuronal cells to test for responsiveness of the neuronal cells to the test compound. Contacting may be in vivo, for example by administering a neurological agent to a subject with a neuronal cell disorder, such as a neurodegenerative disease.

High Throughput Assays The invention provides methods that are useful for screening large numbers of compounds to identify candidates that have a desired level of neurite promoting activity. The invention also provides methods that are useful for screening large numbers of target cell populations to identify cells that are responsive to certain compounds. The invention also provides methods for screening large numbers of suspected toxins or carcinogens to identify compounds that show undesirable levels of neurite outgrowth promotion or inhibition.

Multiple assays are preferably conducted in a format that allows for high throughput screening. High throughput screens can be configured on any of a number of solid supports that allow for rapid, and/or parallel, and/or automated processing and analysis. Preferred methods and devices involve computer controlled automated devices such as robotic devices (e. g. , automated micropipettors) for delivering solutions (e. g. of compound or reagent solutions) and for mixing, incubating and reading assay outputs. In most embodiments, a computer can be programmed to process the raw data from the assays and process it to provide information readout in a user friendly format, such as one of the formats described herein.

According to the invention, detection assays (single assays or multiple assays) can be performed on a solid phase (e. g. the cells or detection reagents are bound to a solid phase) or in solution. Useful assays and formats include, but are not limited to, fluorescence polarization (in solution, direct or in competition), FlashPlate (PerkinElmer), a homogenous radioactive format, Alpha-Screen (PerkinElmer), a free radical amplified chemoluminence format,

Scintillation Proximity Assay (Amershan) in beads format, scintillation proximity in other formats, Enzyme Fragment Complementation Technology (EFC) (DiscoveRx), molecular beacons (e. g. oligonucleotide molecular beacons), homogenous assay formats using Fluorescence Resonance Energy Transfer (FRET), and LANCE (PerkinElmer). Such assays and formats are particularly useful for high throughput screening.

High throughput screening is preferably used when between 10 and 100, between 100 and 1,000, between 1,000 and 10,000, between 10,000 and 100,000, or more than 100,000 different compounds, different target cell populations, different reaction conditions, or a combination thereof are being assayed.

Examples of high throughput screening formats include, but are not limited to, multi- well plates (e. g. 16,24, 64,96, 384,1536, 3456,8000 or more wells) microfluidic systems, arrays of reaction tubes, chips, and other systems that contain a plurality of reaction chambers.

In one embodiment of the above-described methods, one or more reagents are immobilized on a solid support for use in a detection assay. Illustrative examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling agents such as antibodies, peptides and the like to such solid supports are well known in the art, as described, for example, in D. M. Weir et al. , Handbook of Experimental<BR> Immunology, 4th Ed. , Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986)<BR> and W. D. Jacoby et al. , Meth. Enzym. 34, Academic Press, N. Y. (1974).

Additionally, one or more of the agents of the present invention which is used in one of the above-described methods can be detectably labeled prior to use, for example, through the <BR> <BR> use of radioisotopes, affinity labels (such as biotin, avidin, etc. ), enzymatic labels (such as<BR> horse radish peroxidase, alkaline phosphatase, etc. ), as described herein fluorescent labels<BR> (such as FITC or rhodamine, etc. ), paramagnetic atoms, etc. Procedures for accomplishing<BR> such labeling are well-known in the art (L. A. Sternberger et al. , J. Histochem. Cytochem.<BR> <P>18: 315 (1970); E. A. Bayer et al. , Meth. Enzym. 62: 308 (1979); E. Engval et al. , Immunol.

109: 129 (1972); and J. W. Goding, J. Immunol. Meth. 13: 215 (1976)).

Quality Control

The invention also provides methods and compositions useful for quality control analyses of neurite outgrowth modulating drug (e. g. drugs that increase neurite outgrowth).

The effectiveness of the drug can be assayed using an annexin II expression assay of the invention. In some embodiments, a batch of manufactured drug can be assayed by taking one or more representative aliquots and testing them in an annexin II expression assay. The assay should be a standard assay. The amount of annexin II expression is compared to a minimal or threshold level of annexin II expression associated with a minimal or threshold level of activity that is acceptable for the drug. Similarly, annexin II expression methods can be used to verify that a drug does not have too much activity. This testing can be performed by the manufacturer, distributor, or user (e. g. hospital or other medical center) of the drug. Drug batches that are either below a minimal threshold or above a maximal threshold are discarded or returned to the manufacturer. Assays of the invention can also be used to evaluate the activity of a drug that has been stored for long periods of time or under suboptimal storage conditions (e. g. excessive heat or cold).

Kits Methods and compositions of the invention are ideally suited for a kit format. For example, the present invention provides a compartmentalized kit to receive in close confinement, one or more containers which comprises: a) a first container comprising an annexin II binding reagent; and b) one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound agents from the first container.

As used herein, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Illustrative examples of such containers include, but are not limited to, small glass containers, plastic containers or strips of plastic or paper. Particularly preferred types of containers allow the skilled worker to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross- contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers include, but are not limited to, a container which will accept the test sample, a container which contains one or more of the reagents of the present invention used in the assay, containers which contain wash reagents

(such as phosphate buffered saline, Tris-buffers, etc. ), and containers which contain the reagents used to detect the bound reagent.

The types of detection reagents that can be used in the above described kits include, but are not limited to, labeled secondary agents, or in the alternative, if the primary reagent is labeled, enzymatic or agent binding reagents which are capable of reacting with the labeled reagent. One skilled in the art will readily recognize that reagents of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

The assays described herein can be performed, for example, using pre-packaged kits comprising at least one annexin II binding or detection reagent.

In some embodiments, a kit according to the invention includes components that detect and/or measure annexin antigens in the biological sample. For example, where annexin II proteins are detected and/or measured by enzyme linked immunoabsorbent assay (ELISA), such components may include an antibody directed to epitopes of the annexin proteins which can be used to detect and/or quantitate the level of annexin expression in the sample. The antibody itself may be detectably labeled with a radioactive, flourescent, colorimetric or enzyme label. Alternatively, the kit may contain a labeled secondary antibody.

Reagents The following and reagents and biological materials can be used or incorporated into any of the methods, compositions, or kits described herein.

Vectors and Cell Lines The present invention further includes DNA vectors that contain DNA sequences described herein. In some embodiments, the DNA sequences are complementary to annexin II gene sequences and are useful for nucleic acid hybridization assays. In some embodiments, the DNA sequences encode one or more peptide-based annexin II binding reagents that are useful for annexin II protein detection assays. The vectors may be vectors in which the DNA molecules are functionally linked to control sequences that regulate expression of the corresponding mRNAs and polypeptides. These are preferably plasmids which can be replicated and/or expressed in prokaryotes such as E. coli and/or in eukaryotic systems such as yeasts or mammalian cell lines. These vectors may also be mammalian viral vectors which can

be replicated and/or expressed in eukaryotes such as mammalian cell lines and in the human patient, as"host, "for integration into the cellular genome of the patient and expression as genetic therapy systems. Certain vectors also contain one or more selectable markers.

The invention also includes host organisms transformed with the above vectors.

Expression in prokaryotes and eukaryotes may be carried out using techniques known in the art. In some embodiments, DNA sequences can encode fusion polypeptides or intact, native polypeptides. Fusion proteins may advantageously be produced in large quantities. They are generally more stable than the native polypeptide and are easy to purify. The expression of these fusion proteins can be controlled by normal host DNA sequences.

Antibodies Suitable monoclonal and polyclonal antibodies may be prepared by any of the methods and techniques well known in the art, such as described in, for example, A. M. Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984) and Harlow; Antibodies, Cold Spring Harbor Press, N. Y. (1989). For example, an antibody capable of binding to an annexin II protein can be generated by immunizing an animal with an annexin II polypeptide. Any animal (mouse, rabbit, etc. ) which is known to produce antibodies can be utilized to produce antibodies with the desired specificity and suitable methods for immunization of these animals are well known in the art, including, for example, subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of polypeptide used for immunization will vary based on a number of factors, including the animal which is immunized, the antigenicity of the polypeptide selected, and the site of injection.

The polypeptides used as an immunogen may be modified as appropriate or administered in an adjuvant in order to increase the peptide antigenicity. Suitable methods increasing antigenicity are well known in the art, and include, for example, coupling the antigen with a heterologous protein (such as globulin or p-galactosidase) or through the inclusion of an adjuvant during immunization.

A preferred method of generating monoclonal antibodies comprises removing spleen cells from the immunized animals, fusing these cells with myeloma cells, such as SP2/0-Agl4 myeloma cells, and allowing them to become monoclonal antibody-producing hybridoma cells.

Any one of a number of methods well known in the art may be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al., <BR> <BR> Exp. Cell Res. 175: 109-124 (1988) ; Kishimoto et al. , Proc. Natl. Acad. Sci USA 87: 2244-2248<BR> (1990) ). Hybridomas secreting the desired antibodies are cloned and the class and subclass of the secreted antibodies are determined using procedures known in the art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)).

For polyclonal antibodies, antibody-containing antisera is preferably isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.

The present invention further provides hybrid cell lines which secrete monoclonal antibodies selective for the annexin II proteins. These monoclonal antibodies are capable of specifically binding to annexin II, an annexin II subunit, or a fragment thereof. These monoclonal antibodies can be used for qualitative and/or quantitative measurement of annexin II. The present invention therefore also includes test systems which contain the monoclonal antibodies herein described.

Antibodies may be used as an isolated whole antibody, or can be used as a source for generating antibody fragments which contain the antigen binding site of the antibody.

Examples of such antibody fragments include, but are not limited to the Fv, the F (ab), the F (ab) 2, fragment, as well as single chain antibodies.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, <BR> <BR> W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc. , New<BR> York; Roitt, I. (1991) Essential Immunology, 7th Ed. , Blackwell Scientific Publications, Oxford). The pFc'and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc'region has been enzymatically cleaved, or which has been produced without the pFc'region, designated an F (ab') 2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding

sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of"humanized"antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc'regions to produce a functional antibody.

Various methods known in the art can be used to generate such fragments without undue experimentation. Recombinant techniques are preferred for generating large quantities of antibodies, antibody fragments and single chain antibodies, as described, for example, in Pluckthum, Bio/Technology 10, : 163-167 (1992); Carter et al., Bio/Technology 10: 167-170 (1992); and Mullinax et al. , Biotechniques 12: 864-869 (1992). In addition, recombinant techniques may be used to generate heterobifunctional antibodies.

In general, recombinant production of antibodies, antibody fragments or derivatives thereof, uses mRNA encoding an antibody which is isolated from hybridoma cells that produce the desired antibody. This mRNA is then used as a source for generating a cDNA molecule which encodes the antibody, or a fragment thereof. Once obtained, the cDNA may be amplified and expressed according to known methods in a variety of eukaryotic and prokaryotic hosts.

The present invention further includes derivatives of antibodies (antibody derivatives).

As used herein, an"antibody derivatives"contain an antibody of the present invention, or a fragment thereof, as well as an additional moiety which is not normally a part of the antibody. <BR> <BR> <P>Such moieties may improve the solubility, absorption, biological half-life, etc. , of the antibody, decrease the toxicity of the antibody, eliminate or attenuate any undesirable side effect of the antibody, or serve as a detectable marker of the presence of the antibody. Moieties capable of mediating such effects are well known in the art.

Detectably labeled antibodies constitute a special class of the antibody derivatives of the present invention. An antibody is said to be"detectably labeled"if the antibody, or fragment thereof, is attached to a molecule which is capable of identification, visualization, or localization using known methods. Suitable detectable labels include, but are not limited to, radioisotopic labels, enzyme labels, non-radioactive isotopic labels, fluorescent labels, toxin labels, affinity labels, chemiluminescent labels and nuclear magnetic resonance contrast agents described herein.

The coupling of one or more molecules to antibodies is envisioned to include, but is not limited to, many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding, and complexation The covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent agents are useful in coupling protein molecules to other proteins, peptides or amine functions, etc. For example, the literature is replete with coupling agents such as carbodiimides, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylene diamines. This list is not intended to be exhaustive of the various coupling agents known in the art but, rather, is exemplary of the more common coupling agents.

Radionuclides typically are coupled to an antibody by chelation. For example, in the case of metallic radionuclides, a bifunctional chelator is commonly used to link the isotope to the antibody or other protein of interest. Typically, the chelator is first attached to the antibody, and the chelator-antibody conjugate is contacted with the metallic radioisotope. A number of bifunctional chelators have been developed for this purpose, including the dithylenetriamine pentaacetic acid (DTPA) series of amino acids described in U. S. patents 5,124, 471,5, 286,850 and 5,434, 287, which are incorporated herein by reference. As another

example, hydroxamic acid-based bifunctional chelating agents are described in U. S. patent 5,756, 825, the contents of which are incorporated herein. Another example is the chelating agent termedp-SCN-Bz-HEHA (1,4, 7,10, 13,16-hexaazacyclo-octadecane- <BR> <BR> N, N', N", N"', N"", N""'-hexaacetic acid) (Deal et al. , J. Med. Chem. 42: 2988, 1999), which is an effective chelator of radiometals such as 22sAc.

In yet other embodiments, the antibodies can be chimeric or humanized antibodies. As used herein, the term"chimeric antibody"refers to an antibody, that combines the murine variable or hypervariable regions with the human constant region or constant and variable framework regions. As used herein, the term"humanized antibody"refers to an antibody that retains only the antigen-binding CDRs from the parent antibody in association with human framework regions (see, Waldmann, 1991, Science 252: 1657). Such chimeric or humanized antibodies retaining binding specificity of the murine antibody are expected to have reduced immunogenicity when administered in vivo for diagnostic, prophylactic or therapeutic applications according to the invention.

In certain embodiments, the antibodies are human antibodies. The tenn"human antibody", as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline <BR> <BR> immunoglobulin sequences (e. g. , mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term"human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse have been grafted onto human framework sequences (referred to herein as"humanized antibodies"). Human antibodies directed against PSMA can be generated using transgenic mice carrying parts of the human immune system rather than the mouse system.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e. g., U. S. patents 5,591, 669,5, 598,369, 5,545, 806,5, 545,807, 6,150, 584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e. g. , murine) antibodies. The animals are further modified to contain all or a portion of the

human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e. g. , XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.

Another type of annexin II binding reagents are peptide reagents that are classified as antisense-peptide sequences. Antisense-peptide sequences are short peptides which are specifically designed to bind to a particular amino acid sequence. In general, such antisense peptide agents may be generated using methods known in the art, such as those described, for <BR> <BR> example, in Hurby et al. ,"Application of Synthetic Peptides: Antisense Peptides, "in Synthetic<BR> Peptides, A User's Guide, W. H. Freeman, N. Y. , pp. 289-307 (1992) and Kaspczak et al., Biochemistry 28 : 9230-8 (1989).

An additional class of annexin II binding reagents includes natural ligands of annexin II. Annexin II is a co-receptor for tissue plasminogen activator and plasminogen and is found on a wide variety of cell types including endothelial cells, some tumor cells, monocytes and macrophages and neuronal cells.

Clinical Applications Diagnostic Applications According to the invention, measurements of annexin II levels in vivo or in one or more samples obtained from a subject can be used for the early diagnosis of conditions associated with either insufficient or excessive neurite outgrowth. Moreover, the monitoring and quantitation of annexin II expression levels can be used prognostically to stage the progression of a disease or condition and to evaluate the efficacy of agents used to treat a subject.

Clinical Trials Methods and compositions of the invention can be used to identify candidate compounds for testing in clinical trials in humans or other mammals. Methods and compositions of the invention also can be used to evaluate the effectiveness of a candidate

compound administered to a subject such as a clinical trial patient. Accordingly, the invention includes a method for conducting a clinical trial to evaluate the effectiveness of a candidate compound for the treatment of a condition that requires modulation of neurite outgrowth. In preferred embodiments, the candidate compound was selected in an annexin II assay of the invention. In most embodiments, the desired modulation of neurite outgrowth is an increase in neurite outgrowth. Conditions for which increased neurite outgrowth is desired include neurodenerative diseases or disorders (e. g. age-related neurodegeneration, motor neuron diseases, Amyotrophic Lateral Sclerosis, Alzheimer's disease, Parkinsonian syndromes, Huntington's disease, retinal diseases and others) and nerve injuries (e. g severed or crushed nerves, spinal cord injuries, retinal injuries, nerve injuries associated with burns or exposure to toxic agents including specific neurotoxins, brain injuries associate with trauma or ischemia, stroke, and others). In some embodiments, the desired modulation of neurite outgrowth is a decrease in neurite outgrowth. Conditions for which decreased neurite outgrowth is desired include diseases or disorders characterized by excessive neuronal growth (e. g. tumor cell invasion, angiogenesis, inflammation, virus infection, and others). The amount of neurite outgrowth can be monitored in a clinical trial patient by any of a number of methods including but not limited to a) observing the progress of symptoms associated with the condition, b) directly observing neurite outgrowth in vivo using one or imaging techniques, and c) detecting annexin II in vivo (e. g. detecting annexin II levels and changes in annexin II levels in neuronal tissue of interest). In some embodiments, annexin II levels in vivo can be measured using a detection reagent (e. g. a labeled antibody) such as those described herein. For example, in some embodiments the in vivo binding of a radioactive annexin II antibody can be detected in a PET scan.

Therapeutic Applications The invention provides methods for identifying compounds that are useful to promote neurite outgrowth. Such compounds are useful to treat a number of neurological diseases and injuries, including neurodegenerative diseases, trauma, ischemia, and other diseases and injuries where an increase in neurite outgrowth would be therapeutically relevant. For example, compounds of the invention can be particularly useful to treat spinal cord injuries including severed or otherwise physically damaged spinal cord. The invention is also useful to treat (e. g. prevent, slow, or reverse) neurodegeneration associated with aging.

The present invention therefore provides pharmaceutical compositions comprising a one or more compounds identified using an annexin II assay. These pharmaceutical compositions may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. As used herein, "pharmaceutically acceptable carrier"is intended to mean a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term"parenteral"as used herein refers to modes of administration which include, but are not limited to, intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. One of ordinary skill will recognize that the choice of a particular mode of administration can be made empirically based upon considerations such as the particular disease state being treated ; the type and degree of the response to be achieved; the specific agent or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration and rate of excretion of the agent or composition; the duration of the treatment ; drugs (such as a chemotherapeutic agent) used in combination or coincidental with the specific composition; and like factors well known in the medical arts.

Pharmaceutical compositions of the present invention for parenteral injection may comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Illustrative examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include, but are not limited to, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylceuulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like.

Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of the therapeutic agent or inhibitor, it is desirable to slow the absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or rnicroemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compounds are preferably mixed with at least one pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In

the case of capsules, tablets and pills, the dosage form may also comprise buffering agents as appropriate.

Solid compositions of a similar type may also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient (s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Illustrative examples of embedding compositions which can be used include, but are not limited to, polymeric substances and waxes.

The compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

The agent or inhibitor can also be administered in the form of liposomes. As is known to those skilled in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono-or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to the agent or inhibitor, stabilizers, preservatives, excipients, and the like. Preferred lipids are phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, e. g. , Prescott, ed. , METHODS IN CELL BIOLOGY, Volume XIV, Academic Press, New York, N. Y. (1976), p. 33 et seq.

The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically acceptable compositions, whereby these materials, or their functional derivatives, are combined in a mixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e. g., human serum albumin, are well known in the art. In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more compounds of the present invention.

Additional pharmaceutical methods may be employed to control the duration of action.

Controlled release preparations may be achieved through the use of polymers to complex or absorb the therapeutic agents of the invention. The controlled delivery may be exercised by selecting appropriate macromolecules (such as polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate antibodies into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly (methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

The pharmaceutical formulations of the present invention are prepared, for example, by admixing the active agent with solvents and/or carriers, optionally using emulsifiers and/or dispersants, whilst if water is used as the diluent, organic solvents may be used as solubilizing agents or auxiliary solvents. As described above, the excipients used include, for example, water, pharmaceutically acceptable organic solvents such as paraffins, vegetable oils, mono-or polyfunctional alcohols, carriers such as natural mineral powders, synthetic mineral powders, sugars, emulsifiers and lubricants.

One of ordinary skill will appreciate that effective amounts of the therapeutic compounds can be determined empirically and may be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt, ester or prodrug form. The compound can be administered in compositions in combination with one or more pharmaceutically acceptable excipients. It will be understood that, when administered to a human patient, the total daily usage of the agents and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgement. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the type and degree of the response to be achieved; the specific agent or composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the agent or composition; the duration of the treatment; drugs (such as a chemotherapeutic agent) used in combination or coincidental with the specific composition; and like factors well known in the medical arts.

Techniques of dosage determination are well known in the art. The therapeutically effective dose can be lowered if a compound of the present invention is additionally administered with another compound. As used herein, one compound is said to be additionally administered with a second compound when the administration of the two compounds is in such proximity of time that both compounds can be detected at the same time in the patient's serum.

For example, satisfactory results are obtained by oral administration of therapeutic dosages on the order of from 0.05 to 10 mg/kg/day, preferably 0.1 to 7.5 mg/kg/day, more preferably 0.1 to 2 mg/kg/day, administered once or, in divided doses, 2 to 4 times per day. On administration parenterally, for example by i. v. drip or infusion, dosages on the order of from 0. 01 to 5 mg/kg/day, preferably 0.05 to 1.0 mg/kg/day and more preferably 0.1 to 1.0

mg/kg/day can be used. Suitable daily dosages for patients are thus on the order of from 2.5 to 500 mg p. o. , preferably 5 to 250 mg p. o. , more preferably 5 to 100 mg p. o. , or on the order of<BR> from 0.5 to 250 mg i. v. , preferably 2.5 to 125 mg i. v. and more preferably 2.5 to 50 mg i. v.

Dosaging may also be arranged in a patient specific manner to provide a predetermined concentration of a compound in the blood, as determined by the RIA technique. Thus patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.

According to the invention, compounds for treating neurological conditions or diseases can be formulated or administered using methods that help the compounds cross the blood- brain barrier (BBB). The vertebrate brain and CNS has a unique capillary system unlike that in any other organ in the body. The unique capillary system has morphologic characteristics which make up the blood-brain barrier (BBB). The blood-brain barrier acts as a system-wide cellular membrane that separates the brain interstitial space from the blood.

The unique morphologic characteristics of the brain capillaries that make up the BBB are: (a) epithelial-like high resistance tight junctions which literally cement all endothelia of brain capillaries together, and (b) scanty pinocytosis or transendothelial channels, which are abundant in endothelia of peripheral organs. Due to the unique characteristics of the blood- brain barrier, hydrophilic drugs and peptides that readily gain access to other tissues in the body are barred from entry into the brain or their rates of entry and/or accumulation in the brain are very low.

Various strategies have been developed for introducing those drugs into the brain which otherwise would not cross the blood-brain barrier. Widely used strategies involve invasive procedures where the drug is delivered directly into the brain. One such procedure is the implantation of a catheter into the ventricular system to bypass the blood-brain barrier and deliver the drug directly to the brain. These procedures have been used in the treatment of brain diseases which have a predilection for the meninges, e. g. , leukemic involvement of the brain (US 4,902, 505, incorporated herein in its entirety by reference).

Although invasive procedures for the direct delivery of drugs to the brain ventricles have experienced some success, they are limited in that they may only distribute the drug to superficial areas of the brain tissues, and not to the structures deep within the brain. Further, the invasive procedures are potentially harmful to the patient.

Other approaches to circumventing the blood-brain barrier utilize pharmacologic-based procedures involving drug latentiation or the conversion of hydrophilic drugs into lipid-soluble drugs. The majority of the latentiation approaches involve blocking the hydroxyl, carboxyl and primary amine groups on the drug to make it more lipid-soluble and therefore more easily able to cross the blood-brain barrier.

Another approach to increasing the permeability of the BBB to drugs involves the intra- arterial infusion of hypertonic substances which transiently open the blood-brain barrier to allow passage of hydrophilic drugs. However, hypertonic substances are potentially toxic and may damage the blood-brain barrier.

Peptide compositions of the invention may be administered using chimeric peptides wherein the hydrophilic peptide drug is conjugated to a transportable peptide, capable of crossing the blood-brain barrier by transcytosis at a much higher rate than the hydrophilic peptides alone. Suitable transportable peptides include, but are not limited to, histone, insulin, transferrin, insulin-like growth factor I (IGF-I), insulin-like growth factor Il (IGF-II), basic albumin and prolactin.

Antibodies are another method for delivery of compositions of the invention. For example, an antibody that is reactive with a transferrin receptor present on a brain capillary endothelial cell, can be conjugated to a neuropharmaceutical agent to produce an antibody- neuropharmaceutical agent conjugate (US 5,004, 697 incorporated herein in its entirety by reference). The method is conducted under conditions whereby the antibody binds to the transferrin receptor on the brain capillary endothelial cell and the neuropharmaceutical agent is transferred across the blood brain barrier in a pharmaceutically active form. The uptake or transport of antibodies into the brain can also be greatly increased by cationizing the antibodies to form cationized antibodies having an isoelectric point of between about 8.0 to 11.0 (US 5,527, 527 incorporated herein in its entirety by reference).

A ligand-neuropharmaceutical agent fusion protein is another method useful for delivery of compositions to a host (US 5,977, 307, incorporated herein in its entirety by reference). The ligand is reactive with a brain capillary endothelial cell receptor. The method is conducted under conditions whereby the ligand binds to the receptor on a brain capillary endothelial cell and the neuropharmaceutical agent is transferred across the blood brain barrier in a pharmaceutically active form. In some embodiments, a ligand-neuropharmaceutical agent

fusion protein, which has both ligand binding and neurophannaceutical characteristics, can be produced as a contiguous protein by using genetic engineering techniques. Gene constructs can be prepared comprising DNA encoding the ligand fused to DNA encoding the protein, polypeptide or peptide to be delivered across the blood brain barrier. The ligand coding sequence and the agent coding sequence are inserted in the expression vectors in a suitable manner for proper expression of the desired fusion protein. The gene fusion is expressed as a contiguous protein molecule containing both a ligand portion and a neuropharmaceutical agent portion.

The permeability of the blood brain barrier can be increased by administering a blood brain barrier agonist, for example bradykinin (US 5,112, 596 incorporated herein in its entirety by reference), or polypeptides called receptor mediated permeabilizers (RMP) (US 5, 268, 164 incorporated herein in its entirety by reference). Exogenous molecules can be administered to the host's bloodstream parenterally by subcutaneous, intravenous or intramuscular injection or by absorption through a bodily tissue, such as the digestive tract, the respiratory system or the <BR> <BR> skin. The form in which the molecule is administered (e. g. , capsule, tablet, solution, emulsion) depends, at least in part, on the route by which it is administered. The administration of the exogenous molecule to the host's bloodstream and the intravenous injection of the agonist of blood-brain barrier permeability can occur simultaneously or sequentially in time. For example, a therapeutic drug can be administered orally in tablet form while the intravenous administration of an agonist of blood-brain barrier permeability is given later (e. g. between 30 minutes later and several hours later). This allows time for the drug to be absorbed in the gastrointestinal tract and taken up by the bloodstream before the agonist is given to increase the permeability of the blood-brain barrier to the drug. On the other hand, an agonist of blood- brain barrier permeability (e. g. bradykinin) can be administered before or at the same time as an intravenous injection of a drug. Thus, the term"co administration"is used herein to mean that the agonist of blood-brain barrier and the exogenous molecule will be administered at times that will achieve significant concentrations in the blood for producing the simultaneous effects of increasing the permeability of the blood-brain barrier and allowing the maximum passage of the exogenous molecule from the blood to the cells of the central nervous system.

The administration of the agents of the present invention may be for either prophylactic or therapeutic purpose. When provided prophylactically, the agent is provided in advance of disease symptoms such as any Alzheimer's disease symptoms. The prophylactic

administration of the agent serves to prevent or reduce the rate of onset of symptoms. When provided therapeutically, the agent is provided at (or shortly after) the onset of the appearance of symptoms of actual disease. In some embodiments, the therapeutic administration of the agent serves to reduce the severity and duration of Alzheimer's disease.

The function and advantage of these and other embodiments of the present invention will be more fully understood from the examples described below. The following examples are intended to illustrate the benefits of the present invention, but do not exemplify the full scope of the invention.

EXAMPLES Example 1: This example illustrates an embodiment of PC-12 culture conditions for neurite outgrowth assays. In this example, experimental cell growth and assay conditions include some or all of the following steps.

In step 1, PC-12 cells are plated into a collagen-coated 24-well plate (e. g. catalog number: 354408 available from BD Discovery Labware) at a density of 15,000 cells/cm2 (30,000 cells/well).

In step 2, the cells are grown in DMEM media supplemented with 10% horse serum and 5% fetal bovine serum for 24 hours at 37 °C with 5% C02 and 90% humidity.

In step 3, the media is replaced with DMEM media containing 2% horse serum and 1 % fetal bovine serum (low serum media) containing either 0 ng/ml, 2 ng/ml or 50 ng/ml of nerve growth factor (NGF, available from Boehringer Mannheim, catalog no. 1362348). The negative control wells contain no NGF. The positive control wells contain 50ng/ml NGF. The test wells contain 2ng/ml NGF. The test wells are used for compound screening as follows : i) For initial compound screening, 1.0 u, L of each test compound (lOmM in DMSO) is added to give a final concentration of 15 u. M test compound in a well containing 2ng/ml NGF in low serum media.

ii) In subsequent screening of positive test compounds, each compound is added to a final concentration in the range of 0.15 tM to 50 uM in wells containing 2ng/mL NGF in low serum media.

In step 4, the cells are incubated for 48 hours at 37 °C with 5% CO2 and 90% humidity.

In step 5, the low serum media added above (with or without test compound) is replaced with fresh low serum media containing the same amount (either 0,2 or 50 ng/ml) NGF and no test compound and incubated for 96 hours (using the same incubation conditions as in step 4).

The cells are then assayed for neurite outgrowth and/or expression of one or more markers of neurite outgrowth. Methods to detect and measure neurite outgrowth of neuronal cells are described herein including in the following examples.

Example 2: This example illustrates the positive correlation between annexin II expression and neurite outgrowth.

For the validation experiments described below, PC-12 cells were cultured in duplicate sets of plates in order to measure neurite outgrowth for cells in one set of plates versus annexin II expression for cells in the other set of plates.

Measurement of the NGF titration effect on neurite outgrowth : Cells were initially cultured as described in Example 1, steps 1 and 2.

After 24 hours, the media was replaced with low serum media containing either 0 ng/ml, 2ng/ml, l Ong/ml, 30ng/ml, 50ng/ml, or 100ng/mlNGF.

The cells were incubated for 48 hours at 37 ° C with 5% C02 and 90% humidity.

Neurite outgrowth of the cells was determined and the results are shown in Tables 1 and 2 and Figures 1 and 2.

Measurement of the time course of neurite outgrowth underfixed NGF concentration : Cells were initially cultured as described in Example 1, steps 1 and 2.

After 24 hours, the media was replaced with low serum media containing 50ng/ml NGF.

The cells were incubated for 24,48, 72,96 or 120 hours at 37 °C with 5% C02 and 90% humidity.

Neurite outgrowth of the cells was determined and the results are shown in see Figures 3 and 4.

For each of the validation experiments, manual measurement of neurite outgrowth was performed using morphological scoring techniques on whole cells and measurement of annexin II expression was performed by lysing the cells and detecting annexin II protein using annexin II antibody in standard Western blot procedures.

The comparison of manual measurement of neurite outgrowth to annexin II expression levels shows that annexin II is specifically expressed during neurite outgrowth in PC-12 cells.

The results shown in Tables 1 and 2, which are plotted in Figures 1 and 2 respectively, show that NGF stimulation of annexin II expression is dose dependent and correlates positively with neurite outgrowth.

Table 1. Morphological Scoring of Dose Response. NGF Measurement of neurites in six replicate wells Treatment (X100 = % neurite outgrowth) (ng/ml) Well 1 Well 2 Well 3 Well 4 Well 5 Well 6 2 0. 140 0. 0670 0. 0520 0. 050 0. 0680 0. 0530 10 0. 0850 0. 0510 0. 0920 0. 1250 0. 0780 0. 110 30 0. 1340 0. 0530 0. 080 0. 0820 0. 0970 0. 1030 50 0. 1280 0. 070 0. 0910 0. 1360 0. 0960 0. 140 100 0. 0930 0. 1270 0. 1080 0. 1110 0. 0590 0. 113 Table 2. Western Blot Quantitation of Dose Response. NGF Treatment (ng/ml) Annexin II protein (ng) 0 14. 430000 2 68. 950000 10 283. 340000 30 338. 940000 50 355. 140000 100 370. 360000

The results shown in Tables 3 and 4, which are plotted in Figures 3 and 4 respectively, show that NGF stimulation of annexin II expression is time dependent and correlates positively with neurite outgrowth.

Table 3. Morphological Scoring of Time Course. Treatment Measurement of neurites in three replicate wells Time (hours) (X100 = % neurite outgrowth) Well 1 Well 2 Well 3 24 0. 0580 0. 050 0. 0490 48 0. 190 0. 1550 0. 120 72 0. 1740 0. 2330 0. 2170 96 0. 2510 0. 280 0. 2950 120 0. 620 0. 510 0. 440 Table 4. Western Blot Quantitation of Time Course. Treatment Annexin II Time (hours) Protein (ng) 24 3.00 48 121. 78 72 179. 89 96 208. 34 120 317. 04

Example 3: This example illustrates an embodiment of a colorimetric determination of neurite outgrowth by measurement of annexin II expression using ELISA.

PC-12 cells are cultured as in Example 1.

Media is removed from the cells.

The cells are washed once in PBS and then lysed by addition of lysis buffer, which contains 50 mM Tris-HCl buffer (pH 7.5), 1% triton X-100, ImM EGTA, ImM DTT, and 0. 1 % protease inhibitor cocktail.

Mouse monoclonal antibodies against annexin II (Transduction Laboratories, BD Biosciences, catalog no. 610068) are biotinylated with EZ-Link TM Sulfo-NH-LC-LC-Biotin (Pierce) and coated onto a streptavidin plate (PerkinElmer).

The streptavidin coated plate is blocked with 1 % fatty acid free bovine serum albumin (Sigma) in PBS.

A standard amount of annexin II protein ranging from 25 ng up to 1000 ng in PBS containing 0. 5% fatty acid free BSA or cell lysate diluted 1: 10 in PBS is added to wells of the blocked coated plate and incubated at room temperature for one hour.

Each well is washed 6 times with phosphate buffer saline (PBS) containing 0. 1% Tween 20 (PBST), followed by an incubation with a rabbit anti-annexin II polyclonal antibody (US Biological) diluted 1: 250 in PBST containing 0.5% BSA at room temperature for one hour.

After 6 washes with PBST, a reporter antibody, an anti-rabbit antibody which is labeled with horseradish peroxidase (HRP e. g. available from Cell Signalling Technology) diluted

1: 1000 in PBST containing 0.5% BSA is added to the wells and the plate is incubated for one hour at room temperature.

After incubation, the wells are washed 6 times with PBST and tetra methyl benzidine (Pharmingen) is used as substrate to develop a colorimetric readout.

Example 4: This example illustrates an embodiment of a high throughput screen (HTS) to detect neurite outgrowth using a non-radioactive competition assay.

For this HTS, biotinylated antibody and Europium labeled protein are prepared as follows: a) Annexin II monoclonal antibody (Transduction Laboratories) is biotinlated according to the protocol provided in the EZ-link Sulfo-NHS-LC-LC- Biotin kit (Pierce, catalog no. 21338). Briefly, 500 p1 of antibodies (0.5 mg) is added to 5 111 of (50 pg) of biotinylated reagent in PBS. Incubation is carried out on ice for two hours. The mixture is washed 4 times with PBS (2ml). b) Annexin II protein (US Biologicals, catalog no. A2295-20) is labeled with Europium according to the protocol in the DELFIA Eu-labeling reagent (Perkin Elmer Life sciences, catalog number 1244-302). Briefly, 500 u. g ofannexin II in 250 al of 0.1 M sodium biocarbonate (pH=9.3) is added to 0.2 mg of Europium- labeling reagent. The reaction is carried out overnight at 4 °C. The sample is then loaded on a calibrated G-50 column to separate the free labeling reagent from the annexin II. The fractions containing the annexin II are pooled and washed with 50 mM tris-HCl buffer (pH7.5) containing 0.9% NaCl.

Streptavidin coated 384 well plates (Perkin Elmer Life sciences, Catalog No.

SMP614A) are coated with the biotinylated anti-annexin II antibody at 25ng/well in 50 al PBS for 2 hrs at 37 °C.

The antibody solution is removed and the wells of the plate are washed twice with PBS containing 0.1% Tween (PBST).

The wells are blocked with bovine albumin which is fatty acid free (Sigma, Catalog No. A0281) and 0.5% in PBST.

The blocking solution is removed from the wells and the wells are washed twice with PBST.

A standard curve of Eu-labelled annexin II is generated by adding different amounts of annexin II (diluted 1: 5 with unlabelled annexin) i. e. 400,200, 100,50, 25 and 12.5 ng per well in 501 assay buffer (PBS containing 0.5% BSA and 0.5% triton).

Competition with lysate is performed as follows: a) PC 12 cells are plated at 5,000 cells/well in a 384 well collagen coated plate (e. g. BD Discovery Labware, Catalog No. 354666) and treated with NGF at 2 ng/ml and 50 ng/ml for 5 days (control experiment has no treatment of NGF) b) Cells are lysed in 20 u, l of lysis buffer (50 mM Tris pH 7.4 containing 0.5% triton and 0.1 mM PMSF). c) Lysate (0. 5u. l) is then added to a fixed amount of Eu-labelled annexin (200ng) per well in a total volume of 50 u. l. d) The amount of annexin in the lysate is quantified by comparing with the standard curve described above.

Example 5: This example illustrates an embodiment of an alternative ELISA method (Cell ELISA) that does not include a cell lysis step.

PC-12 cells are plated in one or more 96 well collagen coated plates (BD Biosciences- Biocoat) at a density of 10,000 cells/well in DMEM containing 10% Horse serum and 5% fetal bovine serum, and incudbated for 24 hours at 37 °C with 5% C02 and humidity.

On day two, the media is replaced with 2% horse serum and 1 % fetal bovine serum (low serum media) containing either 0 ng/ml, 2 ng/ml or 50 ng/ml of nerve growth factor (NGF). Test compounds in DMSO are added at 15 uM to media containing 2 ng/ml for 48 hours. Positive test compounds are titrated in the range of 0.15 p. M to 50 u. M. After 48 a hour

incubation, the media containing the test compound is replaced with fresh media containing low serum media with 2 ng/ml NGF for a further 96 hour incubation.

On day five, the cells are washed once with PBS and fixed with 100 pl/well 4% paraformaldehyde (Sigma, Catalog No. P6148) + 0. 3% Triton X-100 for 20 minutes at room temperature (RT).

The fixed cells are then washed once with TBS (Tris buffer Saline, 50 mM TrisHCl, pH 7.5, 0.15 N NaCl) and blocked with SuperBlock (Pierce Chemical, Catalog No. 37535) for 1. 5 hrs at RT.

The fixed cells are then incubated with 100 Ill/well of anti-almexin II monoclonal antibody (Transduction Laboratories, Catalog No. 610068) diluted 1: 250 in Superblock (1: 10) + 0.1 % Triton X-100 overnight at 4 °C.

The fixed cells are then washed 3 times with TBST (TBS plus 0. 1% Triton X-100) for 7 minutes each.

The fixed cells are then incubated with 100 u. l/well of anti-mouse Eu-labeled antibody (Perkin Elmer, Catalog No. AD0124) at 0.5 llg/ml and Hoechst 33258 nuclear stain (Sigma, Catalog No. B2883) at 5 llg/ml diluted in Superblock (1: 10) + 0.1 % Triton X-100 for 1 hour at RT.

The fixed cells are then washed 4 times with TBST for 7 minutes each. On the fourth wash, a Hoechst reading is taken in a fluorescence plate reader (Gemini, Molecular Devices) at Ex: 340 nm and Em: 465 nm.

The wash solution is then removed and 100 u. l DELFIA enhancement solution (Perkin Elmer, Catalog No. 1244-104) is added. Following shaking for 1 minute on a plate reader, a reading is obtained in an LJL analyst using 400 (Eu) dichroic mirror.

Example 6: This example illustrates the positive correlation between annexin II expression and neurite outgrowth in the presence of Y27632 or nicotine (both potentiators of neurite outgrowth).

For the experiments described below, PC-12 cells were cultured as described above and test compounds Y27632 and nicotine were added at different concentrations along with NGF at 2 ng/ml.

Figure 5 shows the neurite promoting effect of Y27632 in the presence of 2 ng/ml NGF. Y27632 is Rho kinase inhibitor and this data shows that other Rho kinase inhibitors can be evaluated using annexin II expression as a biomarker.

Figure 6 shows the relative neurite promoting effects of different concentrations of Y27632 and nicotine at different concentrations in the presence of 2 ng/ml NGF. In this experiment, using the method described in Example 5, it was demonstrated that a Cell ELISA can be used to monitor the neurite promoting effects of compounds in a dose dependent manner (e. g. , dose dependent responses of Y27632 or Nicotine known to promote neurite outgrowth).

Having now described some illustrative embodiments of the invention, it should be apparent to those skilled in the art that the foregoing is merely illustrative and not limiting, having been presented by way of example only. Numerous modifications and other illustrative embodiments are within the scope of one of ordinary skill in the art and are contemplated as falling within the scope of the invention. In particular, although many of the examples presented herein involve specific combinations of method acts or system elements, it should be understood that those acts and those elements may be combined in other ways to accomplish the same objectives. Acts, elements and features discussed only in connection with one embodiment are not intended to be excluded from a similar role in other embodiments.

Further, for the one or more means-plus-function limitations recited in the following claims, the means are not intended to be limited to the means disclosed herein for performing the recited function, but are intended to cover in scope any means, known now or later developed, for performing the recited function. Use of ordinal terms such as"first","second","third", etc. , in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc. , or i), ii), etc. does not by itself

connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention.

Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

All patents and patent publications (including US provisional patent application 60/454,254 filed March 13,2003), references and other publications, including kit protocols that are recited in this application are incorporated in their entirety herein by reference.